Project Summary: Gene activity is modulated by the way genomic DNA is packaged into chromatin ? a process termed ?epigenetics?. Epigenetic controls are disrupted in many, if not most, human diseases. Moreover, emerging ?epigenetic therapies? could potentially correct epigenetic defects. However, current tools for studying chromatin and epigenetic mechanisms are imprecise, hindering progress towards understanding gene and genome regulation. The proposed project will develop novel tools for mapping chromatin and transcription factor interactions with single-molecule precision, thereby establishing entirely new capabilities for functional genomics. Chromatin is made up of histones wrapped by DNA. Histones are subject to many chemical modifications (acetylation, methylation, phosphorylation) whose functions remain poorly understood. In a proof-of-principle study, we captured individual chromatin molecules (nucleosomes) on a surface, probed their modifications with fluorescently-labeled antibodies and Total Internal Reflection Fluorescence (TIRF) microscopy, and sequenced the associated DNA. Based on these encouraging results, we now propose to establish robust experimental systems for detecting multiple histone and DNA modifications concurrently on hundreds of millions of individual nucleosomes. We will then adapt these system for analyzing rare cell types and single cells, and for characterizing combinatorial transcription factor ? regulatory element interactions. In summary, we propose innovative systems for investigating combinatorial chromatin and transcription factor interactions with single-molecule precision and genome-wide coverage. Successful implementation of this technology would transform our ability to study chromatin structure, and hasten progress towards defining the sequences and structures that control our genome in health and disease.